The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRb's ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRb's interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G 1 -to-S-phase transition.

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53 À/À mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53 +/À mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.[Keywords: Cohesion; condensation; chromosome instability; cell cycle; chromatin] Supplemental material is available at http://www.genesdev.org.

Terminally differentiated cell types are needed to live and function in a postmitotic state for a lifetime. Cellular senescence is another type of permanent arrest that blocks the proliferation of cells in response to genotoxic stress. Here we show that the retinoblastoma protein (pRB) uses a mechanism to block DNA replication in senescence that is distinct from its role in permanent cell cycle exit associated with terminal differentiation. Our work demonstrates that a subtle mutation in pRB that cripples its ability to interact with chromatin regulators impairs heterochromatinization and repression of E2F-responsive promoters during senescence. In contrast, terminally differentiated nerve and muscle cells bearing the same mutation fully exit the cell cycle and block E2F-responsive gene expression by a different mechanism. Remarkably, this reveals that pRB recruits chromatin regulators primarily to engage a stress-responsive G 1 arrest program.

fThe retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1 ⌬G ) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1 ⌬G/⌬G mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1⌬G/⌬G MEFs display a magnitude of E2F target gene derepression similar to that of Rb1 ؊/؊ cells, even though Rb1 ⌬G/⌬G cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1 ⌬G/⌬G MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G 1 to arrest proliferation. Some Rb1 ⌬G/⌬G mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.T he retinoblastoma tumor suppressor protein (pRB) has a central role in the regulation of the G 1 -to-S-phase transition. Inactivation of its control over cell cycle progression is one of the most common events in cancer (1). The RB protein is thought to regulate entry into S phase through its ability to repress E2F-dependent transcription (2). In the G 1 phase of the cell cycle, a direct interaction between the large pocket domain of pRB (RBLP) and the transactivation domain of E2Fs blocks transcription and recruits chromatin regulators that maintain the cell in G 1 (3). Activation of cyclin-dependent kinases (CDKs) results in the phosphorylation of pRB and the release of E2F transcription factors (4). Free E2Fs then activate a transcriptional program that drives the cell into S phase (3). This model of pRB regulation of E2F dominates our understanding of G 1 -to-S-phase control. Much of our knowledge of this model was derived from studies using viral oncoproteins encoded by small DNA tumor viruses (5, 6). Of particular note, the human papillomavirus E7 protein has been shown to compete for pRB-E2F interactions to deregulate proliferation (7, 8). However, E7 must also target pRB for degradation in order to induce proliferation (8). Thus, the experimental system that gave rise to the pRB-E2F regulatory axis in cell cycle control also suggests that pRB may engage other growth-suppressing activities beyond E2F regulation. By comparison with the pRB-E2F pathway, we know very little about pRB's non-E2F-dependent growth control mechanisms and their relative contribution to cell cycle regulation and tumor suppressor activities.The minimal growth-suppressive region of pRB...

Transforming growth factor ␤ (TGF-␤) is a crucial mediator of breast development, and loss of TGF-␤-induced growth arrest is a hallmark of breast cancer. TGF-␤ has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-␤ cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1 ⌬L and Rb1 NF ), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-␤ growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-␤ signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-␤ cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-␤ in growth control and mammary gland development.

Consultation with key physicians regarding the proposed percent reduction resulted in a 10% dose reduction for all cases when utilizing these three agents. Implementation of a dose rounding protocol for bevacizumab, trastuzumab, and cetuximab represents a potentially substantial cost savings at this institution.

Despite recent advances in the first-line treatment of multiple myeloma, almost all patients eventually experience relapse with drug-resistant disease. New therapeutic modalities are needed, and to this end, SNS01, a therapeutic nanoparticle, is being investigated for treatment of multiple myeloma. The antitumoral activity of SNS01 is based upon modulation of eukaryotic translation initiation factor 5A (eIF5A), a highly conserved protein that is involved in many cellular processes including proliferation, apoptosis, differentiation and inflammation. eIF5A is regulated by post-translational hypusine modification, and overexpression of hypusination-resistant mutants of eIF5A induces apoptosis in many types of cancer cells. SNS01 is a polyethylenimine (PEI)-based nanoparticle that contains both a B-cell-specific expression plasmid expressing a non-hypusinable mutant of eIF5A and a small interfering RNA (siRNA) which depletes endogenous hypusinated eIF5A. Reducing hypusine-modified eIF5A levels was found to inhibit phosphorylation and activity of ERK MAPK and nuclear factor-κB (NF-κB), and thus sensitize myeloma cells to apoptosis resulting from transfection of a plasmid expressing eIF5A(K50R). SNS01 exhibited significant antitumoral activity in both KAS-6/1 (95% inhibition; P < 0.05) and RPMI 8226 (59% inhibition; P < 0.05) multiple myeloma xenograft models following systemic administration. These results highlight the potential of using this approach as a new therapeutic strategy for multiple myeloma.

The high rates of recurrence and low median survival in many B-cell cancers highlight a need for new targeted therapeutic modalities. In dividing cells, eukaryotic translation initiation factor 5A (eIF5A) is hypusinated and involved in regulation of protein synthesis and proliferation, whereas the non-hypusinated form of eIF5A is a potent inducer of cell death in malignant cells. Here, we demonstrate the potential of modulating eIF5A expression as a novel approach to treating B-cell cancers. SNS01-T is a nonviral polyethylenimine-based nanoparticle, designed to induce apoptosis selectively in B-cell cancers by small interfering RNA–mediated suppression of hypusinated eIF5A and plasmid-based overexpression of a non-hypusinable eIF5A mutant. In this study, we show that SNS01-T is preferentially taken up by malignant B cells, inhibits tumor growth in multiple animal models of B-cell cancers without damaging normal tissues, and synergizes with the current therapies bortezomib and lenalidomide to inhibit tumor progression. The results collectively demonstrate the potential of SNS01-T as a novel therapeutic for treatment of a diverse range of B-cell malignancies.